分子生物学学习资料：MB answers 11

AnswerstoWeaverendofchapterquestions

Chapter11GeneralTranscriptionFactorsinEukaryotes

ListinordertheproteinsthatassembleinvitrotoformaclassIIpreinitiationcomplex.

(a)TFIIDwithhelpfromTFIIA(optionalinvitro)bindstotheTATAboxformingtheDAcomplex,(b)TFIIBbindsnext,(c)TFIIFalongwithRNApolymerasebindstoregionspanningfrom-34to+17.(d)TheremainingfactorsbindinorderofTFIIEandTFIIH,formingtheDABPolFEHpreinitiationcomplex.

DescribeandgivetheresultsofanexperimentthatshowsthatTFIIDisthefundamentalbuildingblockoftheclassIIpreinitiationcomplex.

RefertoFig.11.1ReinbergandcolleaguesperformedgelmobilityshiftassayswithdifferentcombinationsofsubunitsofthepreinitiationcomplexalongwithlabeledDNA.ExperimentswithoutTFIID(lane13,Fig11.1aandlane7,Fig11.1b)resultedinnocomplexformation.

DescribeandgivetheresultsofanexperimentthatshowswhereTFIIDbinds.

RefertoFig.11.2.DNaseIbasedfootprintingshowsthatTFIIDprotects(binds)thesmallregionjustaroundtheTATAboxonboththetemplate(Fig11.2a,DNaseIgel,lanelabeledD)andnon-template(Fig11.2b,DNasesIgel,lanelabeledD)strands.

DescribeandgivetheresultsofanexperimentthatshowsthatTFIIFandpolymeraseIIbindtogether,butneithercanbindindependentlytothepreinitiationcomplex.

RefertoFig.11.1.DNAgelmobilityshiftassaysshowedthatwhenTFIIFwasaddedtotheDABcomplexoftranscriptionfactors,therewasnodetectabledifferencebetweentheresultantcomplexandtheDABcomplex;indicatingthatTFIIFwasnotabletobindtheDNA(Fig11.1comparelanes2and3).However,asincreasingconcentrationsofPolIIwasaddedalongwithTFIIF,newcomplexesappearedsuggestingthatTFIIFandRNApolymerasebindtogethertotheDABcomplex(Fig11.1,lanes3-7).Similarly,reactionscontainingdecreasingamountsofTFIIFinthepresenceofPolIIresultedinincreasingamountsofjusttheDABcomplex(i.e.loweredhigherordercomplex,Fig11.1,lanes8-12).

ShowthedifferencebetweenfootprintscausedbytheDABandDABPolFcomplexes.Whatconclusioncanyoureachbasedonthisdifference?

RefertoFigs11.2and11.3.TheDABcomplexformsontheTATAboxprotectingboththetemplateandnontemplatestrandsoftheDNA,althoughtheDNAatposition+10appearstobecomehypersensitivetoDNaseIcleavage.Overall,DABprotectstheTATAboxregionbetween-17and-42,whereasDABPolFcomplexextendsthisprotectedregionanother34basesonthenontemplatestrand,fromposition-17to~+17.TheextendedregionofprotectionisconsistentwiththelargesizeofPolIIandwithitsbindingtoonestrandoftheDNA.

PresentahypothesisthatexplainsthefactthatsubstitutionofdCsfordTsanddIsfordAs,intheTATAbox(makingaCICIbox)hasnoeffectonTFIIDbinding.Providetherationaleforyourhypothesis.

RefertoFig.11.5.TFIIDbindingtotheTATABoxisdependentuponitsTATAbindingprotein(TBP)subunit.(TFIIDiscomposedofTBPandanumberofTBP-associatedfactors(TAFs)).SinceTBPbindsintheminorgrooveofDNA,changestothestericchemistryinthemajorgrooveshouldhavenoeffectuponTFIIDbinding.ThesubstitutionofdC’sanddIsaltersthestericchemistryofthemajorgroovebuthasnoaffectupontheminorgrooveandsothesubstitutionshadnoeffectonTBP/TFIIDbinding.

WhatshapedoesTBP(TATA-boxBindingProtein)have?WhatisthegeometryofinteractionbetweenTBPandtheTATAbox?

RefertoFig.11.6.ThestructureofTBPintheabsenceofDNAwassaddleshapedwithtwo“stirrups”,suggestingthatTBPsitsonDNA(intheminorgroove)likeasaddlesitsonahorse.ThecrystalstructuresolvedinthepresenceofDNAtemplate,however,revealedthatthecurvedundersideofthesaddleinsteadoffittingneatlyovertheDNA,isroughlyalignedwiththelongaxisoftheDNA.ThecurvatureoftheproteinappearstoforcetheDNAtobendalmost80degrees,openinguptheminorgrooveandalmoststraighteningthehelicaltwistinthatregion.

DescribeandgivetheresultsofanexperimentthatshowsTBPisrequiredfortranscriptionfromallthreeclassesofpromoters.

RefertoFig.11.7.GeneticexperimentswithtemperaturesensitivemutantsoftheTBPgenedemonstratedthatTBPisrequiredfortranscriptionfromallthreeclassesofpromoters.InvitrotranscriptionassaysusingPolI,PolII,andPolIIIdependentpromotersrequiredanactiveTBPfortranscriptiontooccur.

DescribeandgivetheresultsofanexperimentthatshowsthataclassIIpromoterismoreactiveinvitrowithTFIIDthanwithTBP.

RefertoFig.11.10.ClassIIpromoterscontainaTATAboxaswellasInrandDPEelements.WhentranscriptionfromsuchapromoteristestedusinganinvitroreconstitutedtranscriptionsystemcontainingeitherTBPorTFIID,theRNAproductsassayedbyprimerextensionaregreater(i.e.moreintensebandsbyautoradiography)inreactioncontainingTFIIDthanTBPalone(seeFig11.10,lanes5versus6,and7versus8).

DescribeandgivetheresultsofanexperimentthatidentifiestheTAFsthatbindtoaclassIIpromotercontainingaTATAbox,aninitiator,andadownstreamelement.

RefertoFig.11.11.Inordertodeterminewhichsubunits(TAFs)ofTFIIDbindtothepromoter,Tjianandcolleaguesphoto-cross-linkedTFIIDwitharadioactivelylabeledDNAfragmentcontainingtheHsp70promoter.Crosslinkingcovalentlyattachestheproteinsthatareintimatelyassociatedwith(boundto)theDNA.Photocrosslinkingisachievedbyincorporatingthenucleotideanalogue,bromodeoxyuridine(BrdU)intotheDNAandallowingTFIIDtobindtothesequence.IrradiationofboundcomplexeswithUVlightcross-linksprotein(s)totheBrdUintheDNA.TheDNAisthennucleasetreatedandtheproteinsaresubjectedtoSDS.AutoradiographyofthegelderivedfromsuchanexperimentshowedthatTAFII250andTAFII150becamelabeled,implyingthatthesetwoproteinshadbeeninclosecontactwiththelabeledDNA’smajorgroove.

DescribeandgivetheresultsofaDNasefootprintingexperimentthatshowshowthefootprintisexpandedbyTAFII250and150comparedwithTBPalone.

RefertoFig.11.12.DNasefootprintingshowedthatTBPcausedafootprintonlyintheTATAboxwhereastheternarycomplexcausedanadditionalfootprintintheinitiatorsequence.

DrawadiagramofamodelfortheinteractionofTBP(andotherfactors)withaTATA-lessclassIIpromoter.

RefertoFig.11.13(bandc).TBPcannotbindbyitselftoaTATA-lesspromotersinceitinteractsdirectlywiththeTATAsequence.However,sinceitispartofTFIIDandTATA-lesspromoterscontainothersequenceelementsthattheTAFsofTFIIDcanbind,TBPiscarriedalonginanyinteractionswithTATA-lesspromoters.Forexample,elementssuchasinitiatorsanddownstreampromoterelements(DPEs)canbindTAFII250andTAFII150andsecureTFIID(containingTBP)tothepromoter.Alternatively,transcriptionfactorsthatbindtospecificsequenceelements(i.e.GCboxes)andinteractswithTFIIDsubunitswouldbeabletorecruitTFIID(withTBP)toTATA-lesspromoters.

WholegenomeexpressionanalysisindicatesthatyeastTAFII145isrequiredfortranscriptionofonly16%ofyeastgenes,andTAFII17isrequiredfortranscriptionof67%ofyeastgenes.Providearationalefortheseresults.

TAFII17,isnotonlyasubunitofTFIID,butisalsoapartofthetranscriptionadaptercomplexknownasSAGA(namedforthethreeclassesofproteinsincontains:SPTs,AdAs,andGCN5,alongwithitsenzymaticactivity,histoneacetyltransferase).SAGAalsocontainsTBP,anumberofTAFs,andappearstomediatetheeffectsofcertaintranscriptionactivatorproteins.Thus,TAFII17playsaroleinseveralimportanttranscriptionrelatedcomplexes,andisthereforerequiredfortranscriptionofalargersetofgenesthanTAFII145

PresentexamplesofclassIIpreinitiationcomplexeswith:

a)AnalternativeTBP.

TRF1(TBP-relatedfactor1)inDrosophilamelanogasterisanalternativeTBPandstimulatestranscriptionjustlikeTBP.ItbindstoTFIIAandTFIIB,whilehavingitsowngroupofTRF-associatedfactorscallednTAFs.TRF1stimulatestranscriptionoftheDrosophilatudorgenewhichcontainstwodistinctpromoters:adownstreampromoterwithaTATAboxandasecondpromoterabout77bpupstreamofthefirstandhasaTCboxrecognizedbyacomplexincludingTRF1.

b)AmissingTAFII.

TAFII30isfoundonlyinafractionofhumanTFIIDs,anditspresencecorrelateswithitsresponsivenesstoestrogen.

c)NoTBPorTBP-likeprotein.

TBP-freeTAFII-containingcomplex(TFTC)isabletosponsorpreinitiationcomplexformationwithoutanyhelpfromTFIIDorTBP.TFTCandTFIIDhavestrikinglysimilar3-Dstructures.Bothcontainagroovelargeenoughtoacceptadouble-strandedDNAbutTFTChasaprojectionatitstopduetodomain5.TFIIDlacksboththeprojectionandthedomain.

WhataretheapparentrolesofTFIIAandTFIIBintranscription?

WithoutTFIIAandTFIIB,thepreinitiationcomplexcannotformandtranscriptioncannotinitiate.TFIIAapparentlyhelpsTFIIDbindtothepromoter.TFIIBservesasalinkerbetweenTFIIDandTFIIF/polymeraseII.Ithastwodomains,oneofwhichisresponsibleforbindingtoTFIID,theotherforcontinuingtheassemblyofthepreinitiationcomplex.ItisessentialforbindingRNApolymerasebecausethepolymerase-TFIIFcomplexwillbindtotheDABcomplex,butnottotheDAcomplex.TFIIBfocusesthepositioningofthetranscriptionstartsite,firstitachievescoarsepositioningbybindingtoTBPandRNApolymeraseattheTATAbox.Then,upon,DNAunwinding,finepositioningofthestartsiteisachievedbyTFIIB-DNAinteractions.

DrawaroughsketchoftheTBP-TFIIB-PolIIcomplexboundtoDNA,showingonlytherelativepositionsoftheproteins.Howdothesepositionscorrelatewiththeapparentrolesoftheproteins?

RefertoFig.11.17.TFIIBcontainstwodomains.TFIIBc(Carboxy-terminaldomain)interactswithTBPandDNAattheTATAbox,withtheDNAbentbyTBPappearingtowraparoundTFIIBcandthepolymerase.Afterthebend,theDNAextendsstraighttowardsTFIIBN(N-terminaldomain),whichliesneartheactivesiteofthepolymerase.Thus,TFIIB,bybridgingbetweenTBPandRNApolymeraseII,positionsthepolymeraseactivesitetoinitiatetheappropriatedistancedownstreamoftheTATAbox.

DescribeandgivetheresultsofanexperimentthatmappedthesitesonRBP1andRBP2thatareinclosecontactwiththefingerandlinkerregionofTFIIB.

RefertoFig.11.19.Interactionsbetweendomainsofproteinscanbeexaminedusingacombinationofphoto-crosslinkingandhydroxylradicalcleavageanalysis.Hydroxylradicalcleavageanalysisexploitscysteineresiduesthatcanbeintroducedintooneofthe(putatively)interactingproteinsbysitedirectedmutagenesis.Toeachcysteine,aniron-EDTA(Fe-BABE)isattached.Thismoietycangeneratehydroxylradicalsthatcleaveproteinchainswithin15angstromsofthecysteine.Thus,anyportionoftheinteractingproteinwithin15angstromsofthecysteinewillbecleaved.ThefragmentsgeneratedaftercleavagecanbevisualizedbySDSandWesternblotting.Usingsuchaprocedure,ChenandHahn,determinedtheregionsofRpb1andRpb2thatareinclosecontactwiththefingerandlinkerregionsofTFIIB.TheydeterminedthatthefingerandlinkerregionsofTFIIBareclosetogetherinthepreinitiationcomplexandthatthispartofTFIIB(i.e.TFIIBN)contactsRNAPolIIintheprotrusion,wall,clamp,andforkregionsofthepolymerase,neartheactivesite.

DescribeandgivetheresultsofanexperimentthatshowsthatTFIIH,butnottheothergeneraltranscriptionfactors,phosphorylatestheIIAformofRNApolymeraseIItotheIIOform.Inaddition,includedatathatshowthattheothergeneraltranscriptionfactorshelpTFIIHinthistask.

RefertoFigs11.20and11.21.ReinbergandcolleaguesincubatedPolIIAwithvariousmixturesoftranscriptionfactors.TheyincludedradiolabeledATPinthereactionstomeasurephosphorylationofthepolymeraseandanalyzedtheirsamplesbygelmobilityshiftassays.First,theydemonstratedthataddingATPhadnoeffectonthemobilityoftheDAB,DABPolF,orDABPolFEcomplexes.However,withTFIIHandATP,theresultantcomplex(DABPolFEH)displayedagelshiftconsistentwithphosphorylationofPolIIA(Fig11.20).Second,theydemonstratedthatTFIID,B,F,andEwereinsufficienttocausephosphorylationandthatTFIIHaloneissufficient.However,phosphorylationofthepolymerasebyTFIIHisenhancedbythepresenceoftheothertranscriptionfactors,particularlyTFIIE.ThiswasevidentbaseduponatimecourseofradiolabeledATPincorporationintoPolIIA(DBFHPolIIAcomplex)byTFIIHinthepresenceandabsenceofTFIIE(Fig11.21panelsbandc).

19.DescribeandgivetheresultsofanexperimentthatshowsthatTFIIHphosphorylatestheCTDofpolymeraseII.

RefertoFig.11.22.ReinbergandcolleaguescleavedtheCTDfromthephosphorylatedPolIIasubunitusingchymotrypsin(aprotease).TheproductswereseparatedbySDS,andthefragmentcontainingtheradiolabeledphosphatewasdeterminedtobetheCTDofPolII.

20.DescribeanassayforDNAhelicaseandshowhowitcanbeusedtodemonstratethatTFIIHisassociatedwithhelicaseactivity.

RefertoFig.11.23.Helicaseactivitycanbedeterminedessentiallybyexaminingtheunwindingofaradiolabeledstrandfromadoublestrandedsubstrate.Thesubstrateconsistsofasmall(singlestrandradiolabeled)pieceofDNAthathasbeenallowedtohybridizetoitscomplementaryregioninamuchlargerunlabeledsinglestrandedpieceofDNA.Helicaseacitivity,i.e.unwindingofthestrandsresultsinseparationofthesmallandlargesinglestrandedpieces,whichcanbevisualizedbyagarosegelelectrophoresisoftheproducts.Additionofthevariousgeneraltranscriptionfactors(i.e.TFIIH),+/-ATP,tothesubstrateandelectrophoreticanalysisoftheproductscanbeusedtodemonstratethatTFIIHisassociatedwithhelicaseactivity.

21.DescribeaG-lesscassettetranscriptionassayandshowhowitcanbeusedtodemonstratethattheRAD25DNAhelicaseactivityassociatedwithTFIIHisrequiredfortranscriptioninvitro.

RefertoFig.11.24.Therad25-ts24DNAhelicasesubunitmutationresultsinatemperaturesensitivevariantofTFIIH.TheG-lesscassetteisatemplatecontainingayeastTATAelementupstreamofa400bpregionwithnoG’sinthenontemplatestrand.ThistemplatecanbeusedtoproducetranscriptsinthepresenceofATP,CTP,andUTP(butnoGTP)of375and350ntinlength.Transcriptionassaysperformedatvarioustemperaturesusingextractsfromwild-typeandthetemperaturesensitivemutantcells(rad25versusrad25-ts24)demonstratedlowerlevelsoftranscriptionevenatthepermissivetemperatureandcompletelossofproductformationatelevatedtemperaturesinthemutantascomparedtothewild-typerad25extract.

22.DrawaroughdiagramoftheclassIIpreinitiationcomplex,showingtherelativepositionsofthepolymerase,thepromoterDNA,TBP,TFIIB,E,F,andH.Showthedirectionoftranscription.

RefertoFig.11.26

23.DescribeandgivetheresultsofanexperimentthatshowsthatTFIISstimulatestranscriptionelongationbyRNApolymeraseII.

RefertoFig.11.27.ReinbergandRoederformedelongationcomplexesbyincubatingpolymeraseIIwithaDNAtemplateandnucleotides,allowinginitiationtooccur.Heparinwasthenusedtobindfreepolymeraseandblocknewinitiationfromoccurring.TheadditionofTFIISenhancedtherateofRNAsynthesisoverbufferalone(controlreaction)by2-foldwithin~3minandby2.6foldafter~7min.

24.PresentamodelforreversaloftranscriptionarrestbyTFIIS.WhatpartofTFIISparticipatesmostdirectly?How?

RefertoFigs.11.28and11.29.TFIISactivatestheRNAseactivityinRNAPolII,whichcleavesofftheextrudedpartofthenascentRNAandcreatesanew3’-terminusintheenzyme’sactivesite.TFIISconsistofthreedomains,oneofwhichfeaturesazincribbon.ThiszincribbonandinparticulartwoacidicresiduesatitstipparticipatedirectlyinmetalcoordinationattheactivesiteofPolIIthatwouldparticipateinribonucleaseactivity.

25.DescribeandgivetheresultsofanexperimentthatshowsthatTFIISstimulatesproofreadingbyRNApolymeraseII.

RefertoFig.11.30.Hawleyandcolleaguesisolatedunlabeledelongationcomplexesthatwerepausedatavarietyofsitesclosetothepromoter.Next,thecomplexeswere“walked”toadefinedpositioninthepresenceofradioactiveUTPtolabeltheRNAinthecomplexes.ATPorGTPwasthenaddedtoextendtheRNAbyonemorebase,toposition+43.Thebasethatshouldbeincorporatedatthatpositionisanadenine,howeverintheabsenceofadeninebutpresenceofguanine,thepolymerasewillincorporateitalthoughatareducedefficiency.Labeledtranscriptswerethen(a)digestedwithRNaseT1tomeasurethemisincorporationofG,or(b)chasedintofulllengthviatheadditionofallfournucleotides,thencleaved(withRNaseT1)tomeasurethelossofGfromposition+43byproofreading.ComparisonofremovalofmisincorporatedG(i.e.proofreading)intheabsenceandpresenceofTFIISrevealedstimulationofproofreadingbyTFIISsuchthatalmostnomisincorporatedproductscouldbedetected.

26.WhatisthemeaningofthetermRNApolymeraseIIholoenzyme?HowdoestheholoenzymedifferfromthecorepolymeraseII?

TheRNApolymeraseIIholoenzymeisacomplexcontainingRNApolymeraseIIandasubsetofgeneraltranscriptionfactorsalongwithotherproteinsthathelpassembleclassIIpreinitiationcomplexes,itincludesthecorepolymeraseII.

27.DescribeandgivetheresultsofanexperimentthatshowstheeffectofaddingorremovingafewbasepairsbetweenthecoreelementandthetranscriptionstartsiteinaclassIpromoter.

RefertoFig.11.31.Paulandcolleaguesmadeinsertionsanddeletionsofupto5bpbetweentheTIF-IBbindingsiteandthenormaltranscriptionstartsiteofarRNApromoter.Transcriptionstilloccurred;however,theinitiationsitemovedupstreamordownstreamaccordingtothenumberofbasepairsaddedordeleted.Additionorsubtractionofmorethan5bp,however,blockedtranscriptionalactivity.

28.WhichgeneraltranscriptionfactoristheassemblyfactorinclassIpromoters?Inotherwords,whichbindsfirstandhelpstheotherbind?DescribeaDNasefootprintingexperimentyouwouldperformtoprovethis,andshowidealizedresults,notnecessarilythosethatTjianandcolleaguesactuallyobtained.Makesureyourdiagramsindicateaneffectofbothtranscriptionfactorsonthefootprints.

ClassIpromotersarerecognizedbytwotranscriptionfactors,acorebindingfactorandaUPE-bindingfactor(UBF).Thecore-bindingfactoristhefundamentaltranscriptionfactorrequiredtorecruitRNAPolI.UBFistheassemblyfactor,asithelpsthecore-bindingfactorbindtothecorepromoterelement,althoughthedegreeofrelianceonUBFvariesconsiderablybetweenorganisms.ForfootprintingresultsrefertoFig.11.32.

29.DescribeandgivetheresultsofcopurificationandimmunoprecipitationexperimentsthatshowthatSL1containsTBP.

RefertoFig,11.34.Tjianandco-workersdemonstratedthatSL1iscomposedofTBPandthreeTAFs.First,theypurifiedhumanSL1byseveraldifferentprocedures(heparin-agarosecolumchromatography,glycerolgradientcentrifugation,immunoprecipitationusingTBPantibodies),usinganS1assaytolocatetheSL1activity.Thesamefractions(i.e.purifiedSL1)werethenassayedforthepresenceofTBPbyWesternblotting,confirmingthatTBPcopurifiedand/orimmunoprecipitatedwithSL1.

30.DescribeandgivetheresultsofanexperimentthatidentifiedtheTAFsinSL1.

RefertoFig.11.35HumanSL1wasimmunoprecipitatedwithanti-TBPantibodyandsubjectedtoSDS.TheTAFswerereleasedfromtheimmunoprecipitatewithguanidine-HClandsubjectedtoSDS.Threepolypeptides,withmolecularmassesof110,63,and48kDwereobserved.ComparisonwithstandardsofknownTAFsidentifiedtheSL1componentsasbeingTAFI110,TAFI63,andTAFI48.

31.HowdoweknowthatTFIIIAisnecessaryfortranscriptionof5SrRNAbutnottRNAgenes?

RefertoFig.11.36.Brownandcolleaguesperformedtranscriptionassaysusingextractsfromvariouscellstestedagainsta5SrRNApromoterandatRNApromoterintheabsenceandpresenceofananti-TFIIIAantibody.Theantibodyblockedtranscriptionfromthe5SrRNApromoterbutnotfromthetRNApromoter.Thus,TFIIIAappearstobenecessaryfor5SRNAbutnottRNApromotertranscription.

32.GeiduschekandcolleaguesperformedDNasefootprintingwithpolymeraseIIIplusTFIIIBandCandatRNAgene.Showtheresultstheyobtainedwith:Noaddedprotein;polymeraseandfactors;andpolymerase,factorsandthreeofthefourNTPs.Whatcanyouconcludefromtheseresults?

RefertoFig.11.37(noaddedprotein,lanec,polymerase+factors,lanea,polymerase+factors+fourNTPs,laneb).Thesedataindicatedthatthe(crude)transcriptionfactorpreparationboundboththeinternalpromoterandanupstreamregion(inthetRNAgene).Specifically,thefactorsandpolymerasestronglyprotectedboxBoftheinternalpromoterandtheupstreamregionandweaklyprotectedboxAoftheinternalpromoter.Whenthepolymerase,factors,andthreenucleotideswereadded,thepolymeraseshifteddownstreamandanewregionoverlappingboxAwasprotected.However,theprotectionoftheupstreamregionpersistedevenafterthepolymerasemovedaway.

33.TheclassicalclassIIIgeneshaveinternalpromoters.Nevertheless,TFIIIBandCtogethercauseafootprintinaregionupstreamofthegene’scodingregion.Drawadiagramofthebindingofthesetwofactorsthatexplainstheseobservations.

RefertoFig.11.39.TFIIICrecruitsTFIIIB(alongwithTBP)upstreamoftheinternalTFIIICbindingsite.AfterRNApolymeraserecruitment,andTFIIIBremainsinplace,evenafterthepolymerasemoves(totheright).Thus,DNasefootprintingwithTFIIIB(+TFIIIC)resultsinafootprintinaregionupstreamofthegene’scodingarea(Fig.11.38).

34.DrawadiagramofwhathappenstoTFIIIBandCafterpolymeraseIIIhasbeguntranscribingaclassicalclassIIIgenesuchasatRNAgene.HowdoesthisexplainhownewpolymeraseIIImoleculescancontinuetotranscribethegene,eventhoughfactorsmaynotremainboundtotheinternalpromoter?

RefertoFig.11.39.TFIIICbindsinternalpromoterpromotesbindingofTFIIIB-recruitsRNApolymeraseIII.RNAPolIIIbeginstranscriptionTFIIIBremainsinplaceaspolymerasemovesright(transcribes),itmayormaynotremoveTFIIICfromtheinternalpromoter.Asindicated,TFIIIBremainsinplace,andcanrecruitmorePolIIIforanotherroundoftranscription.

35.DescribeandgivetheresultsofaDNAasefootprintexperimentthatshowsthatTFIIIB+C,butnotTFIIICalone,canprotectaregionupstreamofthetranscriptionstartsiteinatRNAgene.ShowalsowhathappenstothefootprintwhenyoustripoffTFIIICwithheparin.

RefertoFigs.11.37and11.38.GeiduschekandcolleaguesperformedfootprintingexperimentswithalabeledtRNAgeneandcombinationsofpurifiedTFIIIBandC.TFIICaloneprotectedtheinternalpromoter,especiallyboxBbutdidnotprotecttheupstreamregion.Whenbothfactorswerepresent,boththeupstreamregionandinternalpromoterwereprotected.TFIIIBbyitselfdoesnotresultinafootprintindicatingthatitsbindingisdependentonthepresenceofTFIIIC.However,upstreamprotectionbyTFIIIBsurvivesheparintreatment,buttheprotectionofboxesAandB(TFIIICbindingsite)doesnot.Thus,evenifTFIIICisstrippedoff,theTFIIIBfootprintremains.

36.Describeandgivetheresultsofanexperimentthatshowsthefollowing:OnceTFIIIBbindstoaclassicalclassIIIgene,itcansupportmultipleroundsoftranscription,evenafterTFIIIC(orCandA)arestrippedoffthepromoter.

RefertoFig.11.40.Briefly,complexesformedbyrecruitmentofTFIIIBbyTFIIIC/AwerestrippedofTFIIIC/Abyhighsaltorheparin.Thestrippedtemplateswerepurifiedawayfromtheremoved/unboundfactorsandtestedfortheirabilitytosupporttranscription,whichtheycould.

37.DescribeandgivetheresultsofanexperimentthatdemonstratestheflexibilityofTFIIICinbindingtoboxesAandBthatareclosetogetherorfarapartinaclassIIIpromoter.

RefertoFig.11.41.SentenacandcolleaguesboundyeastTFIIICtoclonedtRNAgeneshavingvariabledistancesbetweentheirboxesAandB.Thecomplexeswerethenvisualizedviascanningtransmissionelectronmicroscopy.WhenthedistancebetweenboxesAandBwaszero,TFIIICappearedasalargeblobonDNA.However,withincreasingdistancebetweenboxesAandB(upto74bpapart,Fig.11.41,paneld),TFIIICappearedastwoglobulardomainsseparatedbyalinkerofincreasinglengthbetweenthem.ThecombinationoflargesizeandstretchabilityallowsTFIIICtocontacttwowidelyseparatedpromoterregionswithitstwoglobulardomains.

38.DiagramthepreinitiationcomplexeswithallthreeclassesofTATA-lesspromoters.Identifytheassemblyfactorsineachcase.

RefertoFig.11.42.

AnalyticalQuestions

Anassemblyfactorshouldbeabletobindindependentlytothepromoterandfacilitatethebindingoftheotherfactor.Toprobethisquestion,youcouldperformagelmobilityshiftexperimentwitheachofthegeneraltranscriptionfactorsindependently,andbothtogether.Let’simaginethatfactor#1isanassemblyfactor.Inthatcase,thegelmobilityshiftexperimentshouldshowashiftofashort,labeledpieceofDNAcontainingthepromoterinthepresenceoffactor#1.Factor#2shouldnotbindbyitself,sothereshouldbenomobilityshiftinthepresenceoffactor#2alone.Butfactor#1shouldallowfactor#2tobind,sotherewillbeasupershiftwhenbothfactorsareaddedtogether.ADNasefootprintingexperimentcouldalsobedonetoanswerthesamequestion.Factor#1shouldmakeafootprintinthepromoterDNA,butfactor#2byitselfshouldnot.However,Factors#1and#2shouldproducealargerfootprintthanfactor#1alone.Nitrocellulosefilterbindingexperimentswouldalsowork,buttheyarenotmuchusedanymore,perhapsbecauseofagr